ABSTRACT

We have investigated the effect of pinealectomy of ewes in pregnancy on the presence of the diurnal rhythm in fetal and maternal plasma concentrations of melatonin. Six ewes were pinealectomized between 104 and 118 days of gestation. Fetal and maternal blood samples were collected during 24-h periods between 125 and 140 days of gestation in the pinealectomized ewes and in an intact control (n = 4). There was a significant diurnal rhythm in both fetal and maternal plasma concentrations of melatonin in the control group. In this group, the fetal and maternal plasma melatonin concentrations were significantly higher in the dark (128·4±6·2 and 192·2± 10·7 pmol/l respectively) than in the light (46·2 ± 4·2 and 25·8 ± 2·1 pmol/l respectively). However there was no diurnal rhythm in either the fetal or maternal plasma melatonin concentrations in the pinealectomized group between 125 and 140 days of gestation. In contrast to the control animals, there was also no light–dark difference in the fetal or maternal plasma melatonin concentrations in four pinealectomized animals sampled frequently in the 3–7 days preceding delivery (mean length of gestation 146·5 days). However, in the pinealectomized sheep there was a gradual increase in the combined light–dark fetal plasma melatonin concentrations during late gestation from 27·9 ± 2·8 pmol/l (at 15–20 days before delivery) to 95·2± 14·1 pmol/l on the day of delivery.

We have therefore demonstrated that the maternal pineal is the major source of the diurnal rhythm in maternal and fetal plasma melatonin concentrations. However maternal pinealectomy does not appear to remove all the melatonin immunoreactivity from the maternal and fetal plasma in late gestation.

Journal of Endocrinology (1989) 120, 459–464

ABSTRACT

We have monitored the 24-h profiles of plasma melatonin concentrations between birth and 10 weeks of age, in lambs which were delivered to, and suckled, either pineal-intact (control group) or pinealectomized (pinealectomized group) ewes. Between 0 and 2 weeks of age, plasma concentrations of melatonin in lambs suckling either intact or pinealectomized ewes were highest at 01.00 h. At this age, however, there was no significant difference in the mean plasma concentrations of melatonin between the entire dark and light phases in lambs in either the control group (dark, 39·7 ± 6·0 (s.e.m.) pmol/l; light, 39·5 ± 8·1 pmol/l) or the pinealectomized group (dark, 79·8 ± 43·3 pmol/l; light, 60·9 ±8·7 pmol/l). Between 3 and 4 weeks of age, however, a diurnal rhythm in plasma melatonin concentrations was clearly present in the lambs in both the control and pinealectomized groups (control group: dark, 164·1 ± 5·6 pmol/l; light 26·2 ± 2·5 pmol/l; pinealectomized group: dark, 52·7± 8·0 pmol/l; light, 19·1 ± 5·3 pmol/l; P<0·001). Between 3 and 10 weeks of age, plasma concentrations of melatonin in the dark phase were significantly (P<0·05) lower in the lambs suckling pinealectomized ewes than in the control group. In both the control lambs and lambs suckling pinealectomized ewes, the mean plasma concentrations of melatonin in the dark and light phases increased significantly (P<0·05) between 3 and 6 weeks after birth.

In conclusion, we have demonstrated that a clear diurnal plasma rhythm in melatonin concentrations does not emerge until 3–4 weeks of age in lambs suckling either pinealectomized or intact ewes. We have also measured lower melatonin concentrations during the dark phase in lambs suckling pinealectomized ewes when compared with lambs suckling pineal-intact ewes between 3 and 10 weeks of age.

Journal of Endocrinology (1990) 125, 97–102

ABSTRACT

We have investigated the effect of fetal hypophysectomy on the localization of dopamine B-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and enkephalin-containing peptides in the fetal sheep adrenal, using immunocytochemical techniques.

Staining with anti-DBH was observed throughout the adrenal medulla in the intact (140–146 days of gestation) and hypophysectomized fetal sheep (147–164 days of gestation) and the newborn lamb (10–12 days after birth). In the adrenal medulla of the lategestation intact fetal sheep and newborn lamb, positive staining with anti-PNMT was observed in the peripheral rim of medullary cells adjacent to the adrenal cortex. After hypophysectomy, there was intense positive staining with anti-PNMT in the peripheral adrenal medullary cells and a small and variable proportion of central adrenal medullary cells were stained with anti-PNMT. In the adrenal gland of the intact fetal sheep and the newborn lamb, there was intense staining with anti-enkephalin in the peripheral rim of adrenal medullary cells. Staining with antienkephalin was less intense in the central medullary cells of the adrenal gland of the intact fetal sheep and the 10- to 12-day-old newborn lamb, and many unstained central medullary cells were present. After hypophysectomy, intense positive staining with antienkephalin was observed throughout the entire fetal adrenal medulla. Therefore, the fetal pituitary, either directly or indirectly through the adrenal cortex, plays a role in regulating the pattern of localization of both PNMT and enkephalin in the fetal sheep adrenal.

Journal of Endocrinology (1989) 121, 425–430

ABSTRACT

We have investigated the possible role of the fetal pituitary and ACTH in the control of the synthesis and post-translational processing of the enkephalin precursor, proenkephalin A (proEnk A), in the fetal sheep adrenal gland in late gestation.

Fetal hypophysectomy (n = 8) or sham operations (n = 4) were performed between 109 and 118 days of gestation. At 138–139 days, either ACTH(1–24) (10·5 μg/0·24 ml saline per h, n = 4) was infused intravenously for 72 h into hypophysectomized fetal sheep or 0·9% (w/v) NaCl alone (0·24 ml/h, n = 4) was infused for 72 h into hypophysectomized fetal sheep and sham-operated animals. At the end of the infusion the pregnant ewe was killed and left or right adrenal glands (n = 12) were collected from the fetal sheep that were intact and given saline (Intact + sal; n = 4), hypophysectomized and given saline (Hx + sal; n = 4) and hypophysectomized and given ACTH (Hx + ACTH; n = 4). Each adrenal was homogenized in acid (acetic acid (1 mol/l)/HCl (20 mmol/l)/2-mercaptoethanol (0·2%)). After centrifugation, the supernatant was loaded onto a Sephadex G-75 column (2·0 × 50 cm), eluted at 80 ml/24 h and fractions were collected (5 ml, n = 42). An aliquot of each fraction (2 ml) was dried down prior to enzymatic digestion (trypsin/carboxypeptidase B) and oxidation with H₂O₂, and assay for methionine-O-enkephalin (immunoreactive Met-O-Enk).

The total adrenal content of immunoreactive Met-O-Enk was significantly greater in the Hx + ACTH group (326·2 ±66·7 (s.e.m.)ng/adrenal) when compared with either the Intact + sal group (152·7 ±44·0 ng/adrenal) or the Hx + sal group (112·1 ±20·8 ng/adrenal). In the adrenal glands from all fetuses immunoreactive Met-O-Enk was found in four molecular weight ranges: < 12 kDa, 12–7 kDa, 7–3 kDa and < 3 kDa. There was no significant difference between the Hx + sal and Hx + ACTH groups in the proportion of immunoreactive Met-O-Enk present in each of the molecular weight ranges in the adrenals and therefore the data from these groups were combined for further statistical analysis. The proportion of immunoreactive Met-O-Enk in the > 12 kDa range was significantly less in the Intact + sal group (5·5 ±2·3%) when compared with the hypophysectomized sheep with or without ACTH replacement (18·7 ± 4·5%).

These data demonstrate that fetal hypophysectomy alters the molecular weight profile of Enk-containing peptides in the adrenal of the fetal sheep and whilst ACTH replacement in the hypophysectomized fetus does not alter the post-translational processing of the Enk-containing peptides, it stimulates an increase in the total amount of immunoreactive Met-O-Enk in the fetal adrenal in late gestation.

Journal of Endocrinology (1992) 134, 369–375

ABSTRACT

We have measured fetal and maternal plasma concentrations of cortisol, prolactin, GH and glucose in samples collected during a 24-h period in 14 animals between 127 and 142 days of gestation. There was a significant increase in both the mean daily plasma cortisol concentration and mean daily coefficient of variation (C.V.) of plasma cortisol concentrations after 135 days of gestation. There was also a significant variation in the fetal plasma cortisol concentrations with a peak occurring at 19.00 h. There was a significant sinusoidal diurnal rhythm in the plasma prolactin concentrations in both the fetal sheep and pregnant ewe and the maximal prolactin concentrations occurred between 19.00 and 23.00 h (fetal) and 21.00 and 01.00 h (maternal). Although no significant diurnal variation was detected in fetal plasma GH concentrations, there was a significant sinusoidal diurnal rhythm in the plasma GH concentrations of the pregnant ewe and the maximal maternal GH concentrations occurred between 21.00 and 01.00 h. Both the fetal and maternal plasma glucose concentrations showed a significant sinusoidal diurnal rhythm. The maximal maternal and fetal glucose concentrations were measured between 21.00 and 01.00 h and between 23.00 and 03.00 h respectively. We have therefore established that diurnal variations in plasma cortisol and prolactin concentrations exist prenatally. Whether the presence of such hormonal rhythms reflects the activity of an endogenous fetal circadian pacemaker remains to be established.

J. Endocr. (1987) 114, 65–72

Leptin mRNA was measured in adipose tissue of fetal sheep by reverse transcription polymerase chain reaction (RTPCR). Abundance of leptin mRNA relative to b-actin mRNA in fetal perirenal adipose tissue increased (P<0.02) with gestation, being higher at 144 d (0.73 +/- 0. 10, n=5) than at 90-91 d (0.40 +/- 0.08, n=6) or 125 d (0.40 +/- 0. 04, n=5) gestation (term approximately 147- 150 d). There was a positive relationship between relative abundance of leptin mRNA (y) and fetal body weight (x)between 90 and 144 d gestation (r 2 =0.27, P<0.01). The slope of the linear dependence of leptin mRNA on fetal weight was 15-fold greater (P<0.001) at 90-91d (y = 2.81x - 1.1, n=6, r 2 =0.71, P<0.025) than between 125-144 d gestation (y = 0.195x - 0.15, n=16, r 2 =0.39, P<0.01). Thus the leptin synthetic capacity of fetal adipose tissue appears to increase in late gestation but this is accompanied by constraint of its sensitivity to fetal body weight. We hypothesise that leptin synthesis in fetal adipose tissue is related to fetal nutrient supply and growth rate.

Abstract

We have used a perifusion system and slices of the anterior pituitary of the fetal sheep combined with specific immunoradiometric assays to investigate the effect of increasing gestational age and cortisol infusion on the output of ACTH(1–39) and the ACTH precursors, proACTH and pro-opiomelanocortin, from the fetal sheep pituitary. Two slices from each fetal anterior pituitary at 106–113 days (n=3), 120–136 days (n=5) and 140–143 days (n=5) of gestation were used. Slices from each anterior pituitary were perifused with the perifusion buffer for at least 120 min prior to the infusion of cortisol (100 nm) for 30 min or buffer alone (control). The anterior pituitary output (fmol/5 min per mg pituitary) of ACTH(1–39) and the ACTH precursors were measured using specific immunoradiometric assays. There was a significant increase in the anterior pituitary secretion rate of ACTH(1–39) between 120 and 136 days (1·04 ±0·23 fmol/5 min per mg) and between 140 and 143 days of gestation (3·08 ±0·33 fmol/5 min per mg). In contrast, there was no change in the secretory rate of the ACTH precursors between 105 and 143 days of gestation. The ratio of the anterior pituitary output of the ACTH precursors:ACTH(1–39) therefore decreased between 120 and 143 of days gestation from 19·10 ±2·05 to 6·36 ± 0·58. There was no effect of cortisol infusion on the anterior pituitary secretion of either ACTH(1–39) or the ACTH precursors before 116 days of gestation. After 120 days, the anterior pituitary output of ACTH(1–39) was significantly decreased by cortisol with the maximal change (43 ± 7%) occurring 10–15 min after the start of cortisol inclusion in the perifusate. Cortisol also altered the secretion of ACTH precursors. Although there was no significant effect with respect to baseline secretion rates, precursor secretion was elevated at the beginning of perifusion with cortisol, compared with precursor secretion after cortisol. The ratio of the anterior pituitary output of ACTH precursors:ACTH(1–39) increased from basal values of 16 ±4 and 12 ±4 (precortisol infusion) to 48 ± 14 at 15 min after the start and 40 ± 14 at 45 min after the end of the cortisol infusion. The differential effects of increasing gestational age and cortisol infusion on the output of ACTH(1–39) and the ACTH precursors may be explained by a change in the functional populations of corticotrophs in the fetal sheep anterior pituitary. These changes may be important in the stimulation of the fetal adrenal cortex which occurs before delivery.

Journal of Endocrinology (1995) 144, 569–576

Nuffield Institute for Medical Research and Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford, 0X3 9DU

(Received 6 April 1978)

Growth hormone (GH) has been located in the ovine foetal pituitary gland by day 50 of gestation (Stokes & Boda, 1968). The concentration of GH in the plasma of foetal sheep is ten times higher than the postnatal value, increasing from 40 ng/ml on day 100 of gestation to 100–120 ng/ml on day 140 (Bassett, Thorburn & Wallace, 1970). After foetal hypophysectomy, the concentration of GH falls to < 2 ng/ml, indicating that it originates in the foetal pituitary gland (Wallace, Stacey & Thorburn, 1973). Labelled GH does not cross the ovine placenta (Wallace et al. 1973). After sectioning the foetal pituitary stalk, the concentration of GH in the foetal plasma drops to approximately 5 ng/ml (Wallace et al. 1973), which implies that the secretion of GH

The concentration of prolactin in samples of fetal plasma and amniotic fluid collected from sheep with indwelling catheters has been measured by radioimmunoassay. There was considerable variation between animals in the concentration of prolactin in fetal plasma which ranged from <2 μg/l to >50 μg/l. In five out of eight fetuses which were sampled at intervals throughout the last 30 days of pregnancy, there was an increase in the concentration of prolactin in the plasma before parturition. The concentration of prolactin in amniotic fluid was consistently low (approximately 1 μg/l). Induction of premature parturition by intrafetal infusion of dexamethasone was accompanied by an increase in the concentration of prolactin in only one of three fetuses infused.

These results indicated that prolactin is present in fetal plasma but that an increase in prolactin in fetal plasma before the onset of parturition does not always occur. The low concentration of prolactin in amniotic fluid contrasts with the high concentrations of this hormone found in the amniotic fluid of the primate.

ABSTRACT

An immunocytochemical staining technique was used to investigate the development of the sheep adrenal medullary cells containing enkephalins and the catecholamine synthetic enzymes dopamine β-hydroxylase (DBH) and phenylethanolamine N-methyl transferase (PNMT). No staining was observed in the adrenocortical cells with any of the antisera used in this study. Positive staining with anti-DBH was observed throughout the medulla in both adult and fetal adrenal glands from 80 days of gestation. Positive staining with anti-PNMT was observed in all glands from as early as 80 days of gestation, and staining with this antiserum was mainly confined to the peripheral medullary cells, which were adjacent to, and interdigitated between, the cells of the adrenal cortex. In the fetus between 80 and 120 days of gestation, staining for the enkephalins was observed in both the peripheral columnar and the central polygonal adrenal medullary cells. After 125 days of gestation and in the adult ewe, the peripheral columnar cells were uniformly stained with anti-enkephalin whereas many unstained cells were present in the central medullary region. Therefore, enkephalin-containing peptides are present in the catecholamine cells of the fetal and adult sheep adrenal and there appears to be a changing pattern in the distribution of the enkephalins in the fetal adrenal in late gestation.

J. Endocr. (1988) 118, 221–226